The proposed research involves both genetic and biochemical studies on genetic regulatory mechanisms in the qa system of the eucaryote Neurospora crassa. The qa system comprises a cluster of one regulatory gene (qa-1) plus three structural genes which encode three inducible enzymes catalyzing sequential steps in the catabolism of quinic acid. The product of the regulatory gene is a protein which appears to act in a positive fashion as an activator in combination with the inducer, quinic acid, to initiate synthesis of the qa enzymes. Genetic studies will be continued in order to characterize as completely as possible the organization and functioning of the qa cluster. Experiments will be undertaken to determine the amino acid sequence of the qa-2 gene product - catabolic dehydroquinase - from wild type and various qa mutants. This information should provide one direct test of the present hypothesis that the qa-1 gene product acts only as a regulator and is not a necessary structural component of the active enzymes. Additional experiments will be undertaken to examine regulation in vitro by developing cell free transcriptional and translational systems. Various approaches will be utilized. The qa mRNA(s) will be isolated employing immunoadsorption techniques and/or chromatography on poly (dT) cellulose. Proof of appropriate mRNA isolation will come from its translation in one of several available cell-free systems. Neurospora chromatin will be isolated and fractionated in order to obtain and identify the qa-containing DNA fragment (employing DNA/RNA and DNA/DNA hybridization techniques) and to examine the regulation of its transcription and subsequent translation in in vitro systems. The qa-l ion regulatory protein will be isolated in order to test the effect of exogeneously added activator (both with and without quinic acid) on in vitro transcription and translation of the qa system. These experiments should lead to a total dissection of the qa regulatory mechanisms.